Gene fusion technology, the fusion of two or more genes into a single gene, has been widely used as a tool in protein engineering, localization and purification. There are two conceptually different methods of making fusions. The simplest method, end-to-end fusions, has been used almost exclusively. The second method, insertional fusion, comprises the insertion of one gene into the middle of another gene. Insertions can result in a continuous domain being split into a discontinuous domain.
One of the first reports of successful insertion of one protein into another was a study by Ehrmann, et al., Proc. Natl. Acad. Sci. USA 87: 7574-8, who described the insertion of alkaline phosphatase (AP) into the E. coli outer membrane protein MalF, as a tool for studying membrane topology. High levels of alkaline phosphatase activity were obtained in the fusions despite the fact that alkaline phosphatase requires dimerization for activity. Since then, AP has been successfully inserted into a number of integral membrane proteins (see, e.g., Bibi and Beja, 1994, J. Biol. Chem. 269: 19910-5; Cosgriff and Pittard, 1997, J. Bacteriol. 179: 3317-23; Lacatena, et al., 1994, Proc. Natl. Acad. Sci. USA 91: 10521-5; Pi and Pittard, 1996, J. Bacteriol. 178: 2650-5; Pigeon and Silver, 1994, Mol. Microbiol. 14: 871-81).
Other proteins, including green fluorescent protein GFP (Biondi, et al., 1998, Nucleic Acids Res. 26: 4946-4952; Kratz, et al., 1999, Proc. Natl. Acad. Sci. USA 96: Siegel and Isacoff, 1997, Neuron 19: 73541; Siegel and Isacoff, 2000, Methods Enzymol. 327: 249-59), TEM1 β-lactamase (Betton, et al., 1997, Nat. Biotechnology 15: 1276-1279; Collinet, et al., 2000, J. Biol. Chem. 275: 17428-33; Ehrmann, et al., 1990, Proc. Natl. Acad. Sci. USA 87: 7574-8), thioredoxin (Lu, et al., 1995, Biotechnology (N Y) 13: 366-72); dihydrofolate reductase (Collinet, et al., 2000, J. Biol. Chem. 275: 17428-33); FKBP12 (Tucker and Fields, Nat. Biotechnol. 19: 1042-6); estrogen receptor-α (Tucker and Fields, 2000, supra), and β-xylanase (Aÿ, et al., 1998, Proc. Natl. Acad. Sci. USA 95: 6613-6618); have been successfully inserted into other proteins. Such fusions at least partially retain the function of the inserted protein.
Doi, et al., 1999, FEBS Letters 453: 305-307, describe a fusion which comprises an insertion of the β-lactamase inhibiting protein (BLIP) polypeptide into a surface loop of the GFP protein. After several rounds of random mutagenesis, polypeptides were obtained which exhibited increased fluorescence upon bind of a ligand (β-lactamase) to the BLIP polypeptide.
More recently, yeast sensors for ligand binding were constructed by the insertion of FKBP12 and the estrogen receptor-α ligand-binding domain into a rationally chosen site in dihydrofolate reductase (DHFR) (see, e.g., Tucker and Fields, 2001, Nature Biotechnology 19: 1042-1046). The site of insertion was at residue 107, a site previously shown to be one tolerant of bisection (Pelletier, et al., 1998, Proc. Natl. Acad. Sci. USA 95: 12141-12146). The two fragments of DHFR divided at 107 were found to be unable to reassemble to form an active enzyme unless the fragments were fused to domains that dimerized (e.g., such as leucine zippers). Yeast expressing the FKBP12-DHFR or ERα-DHFR fusion proteins had an approximate two-fold increase in growth rate in the presence of their respective ligands (FK106 and estrogen) when DHFR activity limited growth. The fusion proteins were either fortuitously temperature sensitive (ERα-DHFR) or designed to be so by mutation (FKBP12-DHFR) in order that subtle changes in growth could be detected upon addition of the ligand.
Generally, methods for generating fusion molecules have not provided a systematic way to functionally couple protein domains.